Worldwide efforts to determine the complete nucleotide sequence of the human genome as well as the genomes of other organisms will result in a huge catalog of sequence information. The goals of the human genome program are to have the project completed by early in the first decade of the 21st century. A major challenge which follows is to identify the functions of the tens of thousands of open reading frames that will result.
One of the technologies which will be utilized for this is antisense-, ribozyme- or DNAzyme-mediated destruction of the sequences, followed by phenotypic and more detailed physiologic and biochemical analyses of cells in which the target has been destroyed. A major problem confronting all antisense technologies is that of rapid throughput screening of sites accessible to pairing by the antisense DNAs and RNAs.
Several approaches have been described to determine the accessibility of a target RNA molecule to antisense or ribozyme inhibitors. One approach uses an in vitro screening assay applying as many antisense oligodeoxynucleotides (antisense ODNs) as possible. Monia et al., Nature Med. 2:668-675 (1996); Milner et al., Nature Biotechnol. 15:537-541 (1997). Another utilizes random libraries of ODNs. Ho et al., Nucleic Acids Res. 24:1901-1907 (1996)(Ho et al. I); Birikh et al., RNA 3:429-437 (1997); Lima et al., J. Biol. Chem. 272:626-638 (1997). The accessible sites can be monitored by RNase H cleavage. Birikh et al, supra; Ho et al., Nature Biotechnol. 16:59-63 (1998) (Ho et al., II). RNase H catalyzes the hydrolytic cleavage of the phosphodiester backbone of the RNA strand of a DNA-RNA duplex.
A pool of semi-random, chimeric chemically synthesized ODNs have been used to identify accessible sites cleaved by RNase H on an in vitro synthesized RNA target. Primer extension analyses were used to identify these sites in the target molecule. Lima et al., supra. Other approaches for designing antisense targets in RNA are based upon computer assisted folding models for RNA. Several reports have been published on the use of random ribozyme libraries to screen effective cleavage. Campbell et al., RNA 1:598-609 (1995); Lieber et al., Mol. Cell Biol. 15, 540-551 (1995); Vaish et al., Biochemistry 36:6459-6501 (1997).
In vitro approaches which utilize random or semi-random libraries of ODNs and RNase H seem to be more useful than computer simulations. Lima et al., supra. However, use of in vitro synthesized RNA does not provide satisfactory results for predicting the accessibility of antisense ODNs in vivo. Recent observations suggest that annealing interactions of polynucleotides are influenced by RNA-binding proteins. Tsuchihashi et al., Science 267:99-102 (1993); Portman et al., EMBO J. 13:213-221 (1994); Bertrand and Rossi, EMBO J. 13:2904-2912 (1994). It is therefore important to utilize cellular RNA-binding proteins in assays for elucidating RNA target accessibility to antisense, ribozyme or DNAzyme binding.
Our invention provides a method for identifying sites on target endogenous cellular (native) RNAs or in vitro-synthesized RNAs which are accessible to antisense, ribozyme, or DNAzyme binding. The underlying principles are that accurate predictions of the folded state of RNA within a cell are not possible, and that binding of antisense ODNs, ribozymes or DNAzymes to a substrate RNA under physiological conditions can be used to identify sites for antisense-mediated, ribozyme-mediated or DNAzyme-mediated RNA destruction or inhibition.
In our method we incubate native or in vitro-synthesized RNAs with defined antisense ODNs, ribozymes or DNAzymes, or with a random or semi-random ODN, ribozyme or DNAzyme library, under hybridization conditions in a reaction medium which is a cell extract containing endogenous RNA-binding proteins, or which mimics a cell extract due to presence of one or more RNA-binding proteins. Any antisense ODN, ribozyme or DNAzyme which is complementary to an accessible site in the target RNA will hybridize to that site. When defined ODNs or an ODN library is used, RNase H is present during hybridization or is added after hybridization to cleave the RNA where hybridization has occurred. RNase H can be present when ribozymes or DNAzymes are used, but is not required, since the ribozymes and DNAzymes cleave RNA where hybridization has occurred. In a preferred embodiment, we use a random or semi-random ODN library in cell extracts containing endogenous mRNA, RNA-binding proteins and RNase H. Examples of our invention using defined antisense ODNs to target native MRNA in cell extracts are reported in Scherr and Rossi, Nucleic Acids Res. 26(22):5079-5085 (1999) and included as Example 2 below.
Various methods can be used to identify sites on target RNA to which antisense ODNs, ribozymes or DNAzymes have bound and cleavage has occurred. We prefer to use terminal deoxynucleotidyl transferase-dependent polymerase chain reaction (TDPCR) for this purpose. TDPCR is described in Komura and Riggs, Nucleic Acids Res. 26(7):1807-11(1998). In order to take full advantage of TDPCR, we incorporate a reverse transcription step to convert the RNA template to DNA, followed by TDPCR. In our invention, the 3xe2x80x2 termini needed for the TDPCR method are created by reverse transcribing the target RNA of interest with any suitable RNA dependent DNA polymerase (reverse transcriptase). We do this by hybridizing a first ODN primer (P1) to the RNA in a region which is downstream (i.e., in the 5xe2x80x2xe2x86x923xe2x80x2 direction on the RNA molecule) from the portion of the target RNA molecule which is under study. The polymerase in the presence of dNTPs copies the RNA into DNA from the 3xe2x80x2 end of P1 and terminates copying at the site of cleavage created by either an antisense ODN/RNase H, a ribozyme or a DNAzyme. The new DNA molecule (referred to as the first strand DNA) serves as first template for the PCR portion of the TDPCR method.
We then carry out the TDPCR procedure, which involves the following steps. We react the reverse-transcribed DNA with guanosine triphosphate (rGTP) in presence of terminal deoxynucleotidyl transferase (TdT) to add an (rG)2-4 tail on the 3xe2x80x2 termini of the DNA molecules. We then ligate a double-stranded ODN linker having a 3xe2x80x2 C2-4 overhang on one strand which base-pairs with the (rG)2-4 tail. We then add two PCR primers. One primer we call a linker primer (LP) because it is complementary to the strand of the TDPCR linker which is ligated to the (rG)2-4 tail (sometimes referred to as the lower strand) The other primer (P2) can be the same as P1, but preferably it is nested with respect to P1, i.e., it is complementary to the target RNA in a region which is at least partially upstream (i.e. in the 3xe2x80x2xe2x86x925xe2x80x2 direction on the RNA molecule) from the region which is bound by P1, but it is downstream of the portion of the target RNA molecule which is under study. (In other words, the portion of the target RNA molecule which is under study to determine whether it has accessible binding sites is that portion which is upstream of the region that is complementary to P2.) We then carry out PCR in the known manner in presence of a DNA polymerase and dNTPs to amplify DNA segments defined by primers LP and P2.
The amplified product can be captured by any of various known methods and subsequently sequenced on an automated DNA sequencer, providing precise identification of the cleavage site. Once this identity has been determined, defined sequence antisense DNA, ribozymes or DNAzymes can be synthesized for use in vitro or in vivo.
Our invention includes the use of reverse transcription followed by TDPCR to identify sites to which antisense ODNs, ribozymes or DNAzymes have bound, regardless of whether the hybridization/cleavage reactions were carried out in a medium which is a cell extract or which mimics a cell extract.